The autonomic nervous system plays a significant role in the regulation of hepatic metabolism. Activation of hepatic sympathetic nerves increases hepatic glucose production (HGP) and glycogenolysis, and increased HGP largely contributes to the pathogenesis of type 2 diabetes mellitus. Therefore, without a full understanding of the neural circuits involved in the regulation of the liver, there is a barrier to develop strategies to control glucose levels via the brain-liver pathway. The overall long-term goal of this proposal is to elucidate the fundamental relationship between central autonomic control and glucose homeostasis. The paraventricular nucleus (PVN) of the hypothalamus is a critical command center controlling autonomic outflow and, thereby, influencing glucose and energy homeostasis. Since impaired glucose homeostasis in diabetic patients involves central circuits controlling autonomic output, the immediate objective of this proposal is to investigate the contribution of pre-sympathetic, liver-related PVN and ventral brainstem neurons to the maintenance of glycemic status using in vivo and in vitro approaches. Our preliminary observations from diet- induced obese (DIO) mice demonstrate an overall shift toward excitation in liver-related PVN neurons and a diminished suppression of excitatory neurotransmission by insulin in pre-sympathetic ventral brainstem projecting PVN neurons. These observations lead to the central hypothesis that monosynaptic connections between pre-sympathetic, liver-related PVN and ventral brainstem neurons are necessary for the regulation of hepatic carbohydrate metabolism. The proposed studies will define hypothalamic and ventral brainstem circuits involved in the sympathetic control of the liver using retrograde viral tracing and circuit mapping in combination with immunostaining. The electrophysiological studies will determine the cellular properties of liver-related neurons in control and DIO mice. Functional connections between PVN and liver-related ventral brainstem neurons will be revealed with optogenetics. The proposed in vivo studies will determine the contribution of liver-related neurons to glucose homeostasis. Pharmacogenetic stimulation and inhibition of neurons will be used in combination with hyperinsulinemic-euglycemic clamp studies in conscious intact and adrenalectomized mice. Sympathetic activity will be assessed in a variety of organs and tissues. The outcomes of the proposed studies will establish the contribution of hypothalamic and ventral brainstem circuits to glucose homeostasis, advance our knowledge of sympathetic control of the liver, and may provide new strategies for the improvement of glycemic status in diabetic patients via autonomic control.